O18 Novel multiplex immunoassay for the simultaneous and discriminative detection of the main autoimmunity markers in Diabetes Mellitus

Abstract

Introduction: The main autoimmunity markers in Type 1 Diabetes Mellitus (DM1) are those that have specificity towards: glutamate decarboxylase (GADA), insulinomaassociated tyrosine phosphatase 2 (IA-2A) and isoform 8 of the Zinc transporter (ZnT8A). Its detection is essential to support the diagnosis of DM1 and to establish the presence of Latent Autoimmune Diabetes of the Adult (LADA) to guarantee adequate treatment in a timely manner. The reference method for its detection (Radioligand Binding Assay, RBA) is highly sensitive and specific, but expensive, polluting and its execution is limited to centers with infrastructure and personnel authorized by the regulatory entity. Therefore, it is essential to develop alternative methods that facilitate access to diagnosis to a bigger number of patients. Aim: The aim of this work was to develop an immunoassay based on Flow Cytometry (FC) for the combined and discriminative determination of the main DM1 markers: GADA, IA-2A and ZnT8A. Materials and methods: 10 sera from pediatric patients with a recent diagnosis of DM1 and 20 sera from normal human controls (NHC) were tested. A double paratope model was used incubating the samples with polystyrene microspheres of 4, 5 and 7 µm adsorbed with the recombinant antigens Trx-GAD, Trx-IA-2 and Trx-ZnT8, respectively, and the corresponding biotinylated proteins. After an overnight incubation at 4 ºC, the immune complexes formed were detected using streptavidin-Phycoerythrin and were acquired on a flow cytometer. All samples were evaluated in parallel by RBA.

Keywords

• inmunoensayo multiplex
• marcadores de autoinmunidad
• diabetes